ABSTRACT Conventional culture vessels are not designed for physiological oxygen delivery. Both hyperoxia and hypoxia - commonly observed when culturing cells and tissues in regular plasticware- have been linked to reduced cellular function and death. An adequate means to provide oxygenation is also critical for stem cell applications in which the differentiation outcome is dependent on oxygen tension levels. We have addressed this problem by devising a novel culture device, the oxygen sandwich. This simple system is designed to deliver oxygen in a quasi-physiological fashion by means of a basal air-permeable perfluorocarbon-silicone (PFC/Si) membrane. Our long-term goal is to establish this system as a new standard for tissue culture, both for applications in which oxygen diffusion rates become limiting (such as 3D culture) and for those that require precise adjustments of oxygenation to steer stem cell differentiation in the desired direction. We will focus our proof-of- concept work on islet/beta cell biology. This is a rapidly expanding market that includes clinical uses (islet transplantation), active in vitro research on adult/fetal islets of several species, and pre-clinicl stem cell research with the potential to revolutionize the treatment of diabetes within the next 5-10 years. The pertinence of our model choice is highlighted by two well-documented observations: [1] Pancreatic beta cells (the current end product used in clinical therapies for diabetes) are highly sensitive to sub- and super-physiological oxygen concentrations; and [2] Stem cell differentiation into beta cells (the subject of worldwide research to replace cadaveric islets for future clinical uses) is exquisitely dependent on evolving oxygen tensions. Our Phase I studies aimed at demonstrating that the enhanced in vitro survival and function observed in PFC/Si-cultured islets also resulted in better pre-clinical transplantation outcomes using a marginal mass xenotransplantation model (human islets into diabetic nu/nu immunodeficient mice). After the successful completion of these studies, our Phase II proposal is based on the following specific aims: (1) Scaling-up and definition of manufacturing process (QA & QC, sterilization) for mass production of PFC/Si culture devices; and (2) biological testing and reproducibility studies in human islets and embryonic stem cells (hESc). This application benefits from the assembly of first-rate teams with highly complementary expertise. Our Phase I results are strongly supportive of the feasibility of this proposal. Success in our research would fill a widely acknowledged gap in our ability to preserve islet cell function and survival in vitro confirming this system as a potential new standard for beta cell biology and differentiation studies. As such positive outcome might ultimately speed up the applicability of new-generation beta cell replacement therapies, this project is greatly relevant to the mission of the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK). Success in these studies will also provide proof of principle of the superiority of our oxygen enhancing technology for many other cell culture applications.